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Solution structure of the main α-amylase inhibitor from amaranth seeds

(2001) EUROPEAN JOURNAL OF BIOCHEMISTRY. 269(8). p.2379-2389
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Abstract
The most abundant alpha -amylase inhibitor (AAI) present in the seeds of Amaranthus hypochondriacus, a variety of the Mexican crop plant amaranth, is the smallest polypeptide (32 residues) known to inhibit alpha -amylase activity of insect larvae while leaving that of mammals unaffected. In solution, H-1 NMR reveals that AAI isolated from amaranth seeds adopts a major trans (70%) and minor cis (30%) conformation, resulting from slow cis-irans isomerization of the Val15-Pro16 peptide bond. Both solution structures have been determined using 2D H-1-NMR spectroscopy and XPLOR followed by restrained energy refinement in the consistent-valence force field. For the major isomer, a total of 563 distance restraints, including 55 medium-range and 173 long-range ones, were available from the NOESY spectra. This rather large number of constraints from a protein of such a small size results from a compact fold, imposed through three disulfide bridges arranged in a cysteine-knot motif. The structure of the minor cis isomer has also been determined using a smaller constraint set. It reveals a different backbone conformation in the Pro10-Pro20 segment, while preserving the overall global fold. The energy-refined ensemble of the major isomer, consisting of 20 low-energy conformers with an average backbone rmsd of 0.29 +/- 0.19 Angstrom and no violations larger than 0.4 Angstrom, represents a considerable improvement in precision over a previously reported and independently performed calculation on AAI obtained through solid-phase synthesis, which was determined with only half the number of medium-range and long-range restraints reported here, and featured the trans isomer only. The resulting differences in ensemble precision have been quantified locally and globally, indicating that, for regions of the backbone and a good fraction of the side chains, the conformation is better defined in the new solution structure. Structural comparison of the solution structure with the X-ray structure of the inhibitor when bound to its alpha -amylase target in Tenebrio molitor shows that the backbone conformation is only slightly adjusted on complexation, while that of the side chains involved in protein-protein contacts is similar to those present in solution. Therefore, the overall conformation of AAI appears to be predisposed to binding to its target alpha -amylase, confirming the view that it acts as a lid on top of the alpha -amylase active site.
Keywords
Amaranthus hypochondriacus, cis-trans isomerization, NMR, X-ray, alpha-amylase inhibitor, NUCLEAR-MAGNETIC-RESONANCE, DIMENSIONAL NMR-SPECTROSCOPY, COUPLING-CONSTANTS, PROTEIN STRUCTURES, MACROMOLECULAR STRUCTURES, ANGSTROM RESOLUTION, SECONDARY STRUCTURE, DISTANCE GEOMETRY, EXCHANGE, QUALITY

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Chicago
Martins, José, M Enassar, M Willem, J Wieruzeski, G Lippens, and S Wodak. 2001. “Solution Structure of the Main Α-amylase Inhibitor from Amaranth Seeds.” European Journal of Biochemistry 269 (8): 2379–2389.
APA
Martins, J., Enassar, M., Willem, M., Wieruzeski, J., Lippens, G., & Wodak, S. (2001). Solution structure of the main α-amylase inhibitor from amaranth seeds. EUROPEAN JOURNAL OF BIOCHEMISTRY, 269(8), 2379–2389.
Vancouver
1.
Martins J, Enassar M, Willem M, Wieruzeski J, Lippens G, Wodak S. Solution structure of the main α-amylase inhibitor from amaranth seeds. EUROPEAN JOURNAL OF BIOCHEMISTRY. 2001;269(8):2379–89.
MLA
Martins, José, M Enassar, M Willem, et al. “Solution Structure of the Main Α-amylase Inhibitor from Amaranth Seeds.” EUROPEAN JOURNAL OF BIOCHEMISTRY 269.8 (2001): 2379–2389. Print.
@article{138179,
  abstract     = {The most abundant alpha -amylase inhibitor (AAI) present in the seeds of Amaranthus hypochondriacus, a variety of the Mexican crop plant amaranth, is the smallest polypeptide (32 residues) known to inhibit alpha -amylase activity of insect larvae while leaving that of mammals unaffected. In solution, H-1 NMR reveals that AAI isolated from amaranth seeds adopts a major trans (70\%) and minor cis (30\%) conformation, resulting from slow cis-irans isomerization of the Val15-Pro16 peptide bond. Both solution structures have been determined using 2D H-1-NMR spectroscopy and XPLOR followed by restrained energy refinement in the consistent-valence force field. For the major isomer, a total of 563 distance restraints, including 55 medium-range and 173 long-range ones, were available from the NOESY spectra. This rather large number of constraints from a protein of such a small size results from a compact fold, imposed through three disulfide bridges arranged in a cysteine-knot motif. The structure of the minor cis isomer has also been determined using a smaller constraint set. It reveals a different backbone conformation in the Pro10-Pro20 segment, while preserving the overall global fold. The energy-refined ensemble of the major isomer, consisting of 20 low-energy conformers with an average backbone rmsd of 0.29 +/- 0.19 Angstrom and no violations larger than 0.4 Angstrom, represents a considerable improvement in precision over a previously reported and independently performed calculation on AAI obtained through solid-phase synthesis, which was determined with only half the number of medium-range and long-range restraints reported here, and featured the trans isomer only. The resulting differences in ensemble precision have been quantified locally and globally, indicating that, for regions of the backbone and a good fraction of the side chains, the conformation is better defined in the new solution structure. Structural comparison of the solution structure with the X-ray structure of the inhibitor when bound to its alpha -amylase target in Tenebrio molitor shows that the backbone conformation is only slightly adjusted on complexation, while that of the side chains involved in protein-protein contacts is similar to those present in solution. Therefore, the overall conformation of AAI appears to be predisposed to binding to its target alpha -amylase, confirming the view that it acts as a lid on top of the alpha -amylase active site.},
  author       = {Martins, Jos{\'e} and Enassar, M and Willem, M and Wieruzeski, J and Lippens, G and Wodak, S},
  issn         = {0014-2956},
  journal      = {EUROPEAN JOURNAL OF BIOCHEMISTRY},
  keyword      = {Amaranthus hypochondriacus,cis-trans isomerization,NMR,X-ray,alpha-amylase inhibitor,NUCLEAR-MAGNETIC-RESONANCE,DIMENSIONAL NMR-SPECTROSCOPY,COUPLING-CONSTANTS,PROTEIN STRUCTURES,MACROMOLECULAR STRUCTURES,ANGSTROM RESOLUTION,SECONDARY STRUCTURE,DISTANCE GEOMETRY,EXCHANGE,QUALITY},
  language     = {eng},
  number       = {8},
  pages        = {2379--2389},
  title        = {Solution structure of the main \ensuremath{\alpha}-amylase inhibitor from amaranth seeds},
  url          = {http://dx.doi.org/10.1046/j.1432-1327.2001.02118.x},
  volume       = {269},
  year         = {2001},
}

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